Here, we present a facile technique for synthesis of graphene nanosheet (GNS)-grafted double-walled carbon nanotube (DWCNT) hybrid carbon nanostructures (here after referred to as G-DWCNTs) by directly growing GNSs along the sidewalls of DWCNTs using a two-step chemical vapor deposition (CVD). DWCNTs were synthesized by floating catalyst CVD at 1300 °C using ferrocene and thiophene dissolved in ethanol. Then, GNSs were grafted onto the synthesized DWCNT bundles by thermal CVD at 1300 °C using ethanol. The sharp-edged petal-like structure of GNSs were grown along the sidewalls of DWCNT bundles while maintaining the one-dimensional structure of DWCNT. Next, DWCNTs and G-DWCNTs were dispersed in ethanol, then deposited on the paper using vacuum filtration method and used for ethanol detection. G-DWCNTs sensor exhibited a 3-fold improvement in the response to ethanol vapor compared to the DWCNTs sensor. The sensing mechanism of DWCNTs and G-DWCNTs can be described in terms of charge transfer between the gas molecules and sensing material. These results demonstrate that the facile technique by two-step CVD method provides a promising approach for simple and low-cost technique to synthesize the hybrid nanostructure of GNSs and DWCNTs. The new hybrid carbon nanostructures are attractive for gas sensing application. Graphene and CNTs become an attractive material for research area in the field of nanotechnology. In recent years, the combining between CNTs and graphene have attracted increasing attention because hybrid graphene-CNTs structure possesses the outstanding properties of both two-dimensional graphene and one-dimensional CNTs. Graphenated carbon nanotubes (G-CNTs) are one of the promising of graphene-CNTs three-dimensional hybrid nanostructure that consists of petal-like graphene grown on the sidewalls of CNTs. The advantage of G-CNT hybrid nanostructures is the high surface area of three-dimensional structure. In addition, the G-CNTs have the high edge density of graphene which provide higher charge density and reactivity than the basal plane. Due to their unique structure and outstanding properties, G-CNTs have been widely applied in the areas of the supercapacitor13, gas sensor14, electronic and optoelectronic devices14, and proton exchange membrane fuel cells16. To synthesize G-CNTs, microwave plasma-enhanced chemical vapor deposition (MPECVD) has been considered as a promising method. There have been several reports on the synthesis of G-CNTs using one- or two-step MPECVD20. For instance, Yu et al.14 synthesized the G-CNTs by two-steps MPECVD. Multi-walled CNTs (MWCNTs) were grown by the decomposition of acetylene. Then, a few-layer graphene (FLG) were directly grown on the sidewall of MWCNTs using methane. The gas sensor fabricated from MWCNTs-FLG enabled 3-fold improved nitrogen dioxide detection compared with pristine MWCNTs. In addition, the MWCNTs-FLG structure exhibits remarkable optoelectronic property. The one-step MPECVD technique for synthesis of G-CNTs using methane as carbon source was proposed by Henry et al.19. The G-CNTs exhibited the catalytic activity and electron transfer kinetics two orders magnitude higher and faster than that of CNTs. Recently, there is a report of synthesis of G-CNTs by two-step CVD procedure consisting of thermal CVD (TCVD) and radio frequency plasma-enhanced CVD (RF PECVD)16. In the first step, MWCNTs were grown directly onto the catalyzed carbon paper by the TCVD using ethylene. In the second step, the G-CNT hybrid was fabricated by growth of graphene directly onto the CNTs without any additional catalyst by the RF PECVD technique using acetylene. The G-CNT-supported platinum (Pt) catalyst exhibits a superior electrochemical stability over that of the carbon black-supported Pt catalyst due to the high crystallinity of the G-CNT support. Although the MPECVD has been considered as a promising method for the synthesis of G-CNT hybrid nanostructures, this technique requires expensive vacuum apparatuses and plasma systems. Moreover, the surface of CNT may deteriorate during the high-energy plasma process, thus the PECVD method can produce the hybridization of GNSs with only MWCNTs. It is still challenging to achieve a hybridization of GNSs and thin-walled CNTs, i.e., double-walled CNTs (DWCNTs).GrapheneIn this work, we have proposed the facile synthesis of hybrid nanostructures of graphene nanosheet (GNS) grafted double-walled CNT (DWCNT) bundles (hereafter referred to as G-DWCNTs) by two-step CVD method. First, DWCNTs were synthesized by floating catalyst CVD (FCCVD) method, and subsequently GNSs were deposited on the DWCNTs bundles by TCVD method. The structural evolution between GNS and DWCNT bundles are also demonstrated at different deposition times in the TCVD method. The proposed G-DWCNT hybrid nanostructures show figures of merit over the previous reports in terms of the new hybrid carbon nanostructures and simplicity in synthesis method. In addition, synthesized DWCNTs and G-DWCNTs were used as gas sensor for ethanol detection at parts per million (ppm) levels under room temperature. The fabricated sensor from G-DWCNTs enabled an approximately 3-fold improvement in ethanol detection compared to DWCNTs. The improvement in the sensor response of G-DWCNTs is attributed to the GNS-grafted on DWCNT bundles, which provides higher surface area for gas adsorption.22. The thickness of amorphous carbon layer was estimated to be 20–30 nm. The existence of amorphous carbon as an interface layer enables growth of the GNSs without catalyst. These interesting phenomena will be discussed later in terms of growth mechanism. Figure Figure 23. These results implying the high crystallinity of hybrid carbon nanostructures between GNSs and DWCNT bundles. Figure 24. Figure Figure −1, disorder carbon-derived D-band at ∼134 cm−1, graphitic structure-derived G-band at ∼159 cm−1 and second-order of D-band-derived 2D-band at ∼267 cm−1. In addition, the spectrum of G(20)-DWCNTs shows the D’ band at 1620 cm−1, which originate from the intravalley double resonance process only in presence of defects27. The position of D-band, G-band and 2D-band and the intensity ratio between the D- and G-bands (ID/IG) and the 2D- and G-bands (I2D/IG) of DWCNTs and G-DWCNTs are summarized in the Table ID/IG is an indication of the crystallinity28 of DWCNTs and G-DWCNTs. The calculated ID/IG ratio of DWCNTs was 0.03. After GNSs deposition, the ID/IG ratios of G(2.5)-DWCNTs, G(5)-DWCNTs, G(10)-DWCNTs and G(20)-DWCNTs were 0.05, 0.07, 0.11, and 0.37, respectively. The ID/IG ratio of G-DWCNTs increased with increasing the deposition time of GNSs, implying that the purity and crystallinity of G-DWCNTs were deteriorated due to the reconstructed between GNSs and DWCNTs, indicating the hybridization of GNSs and DWCNTs, specially for G(20)-DWCNTs which is consistent with the TEM results with the highest amount of GNSs -DWCNTs, G(5)-DWCNTs, G(10)-DWCNTs and G(20)-DWCNTs were 0.16, 0.18, 0.19, 0.22 and 0.44, respectively. The increase in the I2D/IG ratio suggests the presence of more graphene growth on the DWCNT surface29. Figure RBM = 234/dt + 10, where ωRBM and dt are the Raman shift of RBM peak (cm−1) and tube diameter (nm), respectively30. The calculated diameters of DWCNTs from RBM spectra at 155, 183, and 268 cm−1 were 1.61, 1.35, and 0.91 nm, respectively. The calculated inner and outer diameters of DWCNTs are in the same range as that observed from the TEM results. After GNSs deposition on DWCNT bundles, the RBM intensity of outer diameter significantly decreased with increasing the TCVD reaction time. The decrease in the RBM intensity of outer diameter could be attributed to the higher amount of GNSs deposition on the DWCNT bundles, resulting in a decrease in a vibration mode in the RBM intensity of outer tube.Raman spectroscopy was employed to characterize the structure, purity and crystallinity of DWCNTs and G-DWCNTs. Figure NSs Fig. . The ID/I-V curves of the DWCNT and G(20)-DWCNT sensors with silver contact electrodes. The I-V curves exhibited linear behavior, implying that the contact between DWCNT and G(20)-DWCNT papers and electrodes is Ohmic contact. The initial resistances of DWCNT and G-DWCNT sensors are 13.59 and 46.23 kΩ, respectively. The initial resistance of G-DWCNTs shows a slight increase from that of DWCNTs, which corresponds to the ID/IG value -DWCNT sensor exhibited a 3.2-fold improvement in the response to ethanol vapor compared to the DWCNT sensor. The enhancement of sensor response could be derived from the high surface area of GNS-grafted DWCNTs which provide the area for molecule adsorption. These results are similar to previous work which MWCNTs-FLG sensor enabled 3-fold improvement in nitrogen dioxide detection compared to pristine MWCNTs sensor. Regarding the response time of the sensor, the sensor response time is defined as the time taken to reach 90% of its maximum sensor response. The sensor response of DWCNTs and G-DWCNTs were 480 and 428 s. These results show that the G-DWCNT sensor shows a merit for enhancement and improvement of sensor response magnitude and response time, respectively. The sensing mechanism of DWCNTs and G-DWCNTs can be explained by charge transfer between the adsorbed gas molecules and sensing materials. Upon ethanol adsorption, electron charge transfer from ethanol molecules to DWCNTs and GNSs, decreasing the density of holes in the p-type DWCNTs and p-type GNSs34, resulting in an increase in the electrical resistance.Figure 37:θ is the monolayer coverage of gas molecules on sensor surface, K (ppm−1) is the equilibrium constant for gas adsorption and C (ppm) is gas adsorption concentration. θ is assumed to be proportional to the sensor response, that is, θ = S/Smax where, Smax is the maximum response of the sensor at saturation coverage. Thus, C is given by Eq. -DWCNTs were measured as a function of ethanol concentration (solid squares and circles) in the range of 352–38, 115 ppm as shown in Fig. 0-DWCNTs K values of DWCNTs and G(20)-DWCNTs were estimated to be 7.29 ×10−5 and 1.16 × 10−4 ppm−1, while Smax values of DWCNTs and G(20)-DWCNTs were 3.51 and 6.32, respectively. The results show that the experiment data has a good fitting to Langmuir isotherm fitting curve  of DWCNTs and G(20)-DWCNTs were investigated. 30 data points prior to exposure to ethanol were plotted and fitted by a fifth-order polynomial. The yi is the measured value of the sensor response baseline and y is the corresponding value calculated from the fifth-order polynomial fit. Then, the rmsnoise of the sensor was calculated by Eq. (N is the number of data points used for the curve fitting (N = 30). Finally, the LOD can be derived from Eq. -DWCNTs and DWCNTs and are approximately 13 and 257 ppm, respectively. The LOD of the G(20)-DWCNTs shows an approximately 20-fold higher than that of DWCNTs. We envision that the sensor based on G-DWCNTs could be down part per billion level for ethanol detection by functionalization of G-DWCNTs with metal nanoparticle40.The calculated 20, our technique shows a benefit in terms of easy set-up without a requirement of vacuum system. Furthermore, from the growth mechanism aspects, this facile two-step CVD technique enables the hybrid structures of GNSs with other types of carbon nanostructure, organic and inorganic nanomaterials, providing novel properties emergent at the nanoscale. Furthermore, the synthesized new hybrid G-DWCNTs can be further used as gas sensor for VOC detection.The facile technique by two-step CVD proposed in this research affords an advantage to grow the hybrid graphene and thin CNT such as DWCNTs while maintains its pristine nanostructure. Moreover, compare to the previous reported methods, such as the plasma-enhanced CVDThe hybrid carbon nanostructures consist of GNSs and DWCNT bundles were successfully synthesized by a simple CVD method. DWCNTs were synthesized by FFCVD method at 1300 °C using ferrocene, thiophene and ethanol. Then, GNSs were grown on the carbon layer-deposited DWCNT bundles by TCVD method at 1300 °C method using ethanol. The deposition time in the TCVD method plays an important role on the amount and density of foliated graphene on the sidewalls of DWCNT bundles. The growth mechanism of G-DWCNT hybrid nanostructures could be described by the carbon layer-deposited on the DWCNT bundle, which acts as the intermediate between GNSs and DWCNT bundles. These results demonstrate the promising hybrid carbon nanostructures grown by facile CVD with simplicity and low-cost techniques. In addition, G(20)-DWCNTs exhibited 3-fold improvement in the sensor response to ethanol compared to DWCNTs. The features of sensor response of DWCNTs and G(20)-DWCNTs can be described by the Langmuir adsorption isotherm. The sensing mechanism of DWCNTs and G-DWCNTs can be described in terms charge transfer between the gas molecules and sensing material. These results demonstrate that G-DWCNT hybrid nanostructures can use as gas sensing device for ethanol detection. Furthermore, these structures can be further used in a wide range of application in the areas of electronic and energy-related devices, and so on.2) gas at a flow rate of 1000 sccm served as carrier gas was introduced into CVD system. The reaction was carried out for 20 min at atmospheric pressure. The as-synthesized samples were continuously grown and formed in the reaction zone, and immediately transferred to deposit on the stainless steel mesh at the outlet. Finally, the as-synthesized samples were cooled down to room temperature under Ar atmosphere at a flow rate of 500 sccm. The as-synthesized samples were further purified by acid and air-annealing treatments repeatedly for 2 times. The as-synthesized samples were soaked in 37% hydrochloric (HCl) acid for 24 h, then filtered and washed with distilled water until pH 7. Next, the samples were annealed under air atmosphere at 400 °C for 30 min in order to remove the amorphous carbon and the metal catalysts. Then, the samples were again washed with HCl and, subsequently annealed under air atmosphere at 500 °C for 30 min. Next, the purified DWCNTs were placed at the middle reaction zone of CVD to graft GNSs on DWCNTs using ethanol as carbon source. The temperature was raised up to 1300 °C under Ar gas at a flow rate of 500 sccm. The ethanol solution was injected into reaction zone of CVD system by micropump at a pumping speed of 0.04 mL/min using H2 gas at a flow rate of 1000 sccm. The CVD synthesis was carried out for 2.5–20 min at atmospheric pressure. Finally, the hybrid carbon nanostructures were cooled down to room temperature under Ar atmosphere at a flow rate of 500 sccm.DWCNTs were synthesized by FCCVD method using ferrocene  and thiophene  dissolved in ethanol solution  as metal catalyst and carbon source, respectively. All substances were mixed by sonication for 30 min before using in the synthesis process. A stainless steel mesh was used as substrate, which was placed at the outlet of alumina tube. At the beginning of the reaction, argon (Ar) gas at a flow rate of 500 standard cubic centimeters per minute (sccm) was introduced into CVD system until the temperature was raised up to 1300 °C. Then, the homogenous mixed solution was injected into reaction zone of CVD system by micropump at a pumping speed of 0.04 mL/min. At the same time, hydrogen (HI-V) characteristics were measured in the voltage range of −2 to 2 V by a source measurement unit.The morphologies and nanostructure of the DWCNTs and G-DWCNT hybrid nanostructures were characterized by field emission scanning electron microscopy  and transmission electron microscopy  operated at 10 and 200 kV, respectively. Raman spectroscopy (Horiba Jobin Yvon) was employed to characterize the crystallinity and purity using a wavelength of 532 nm (2.33 eV). The current-voltage . The fabricated sensors from DWCNTs and G(20)-DWCNTs were placed in a stainless steel chamber and then N2 gas was introduced into the chamber at a flow rate of 500 sccm for 5 min as a baseline. Then, ethanol vapor prepared from bubbling liquid ethanol with 5–500 sccm of nitrogen (N2) gas to measure the sensor response of DWCNT and G(20)-DWCNT sensors. The detection was carried out for 10 min. The ethanol concentration is in the range of 352–38, 115 ppm. For recovery, the sensor was recovered by N2 gas at a flow rate of 500 sccm until its initial electrical resistance. The sensor response (S (%)) of all sensors was calculated using the Eq. -DWCNTs were separately sonicated with ethanol at the concentration of 0.01 mg/mL for a designed time period. The dispersions of DWCNTs and G(20)-DWCNTs were separately vacuum filtered by using a polytetrafluoroethylene (PTFE) membrane  and dried at 100 °C for 60 min. The DWCNTs and G(20)-DWCNTs on PTFE were cut in to a size of 1 × 2 cmFigure S1


Ethylene glycol (EG)-based lubricant was prepared with dissolved organosolv lignin from birch wood (BL) and softwood (SL) biomass. The effects of different lignin types on the rheological, thermal, and tribological properties of the lignin/EG lubricants were comprehensively investigated by various characterization techniques. Dissolving organosolv lignin in EG results in outstanding lubricating properties. Specifically, the wear volume of the disc by EG-44BL is only 8.9% of that lubricated by pure EG. The enhanced anti-wear property of the EG/lignin system could be attributed to the formation of a robust lubrication film and the strong adhesion of the lubricant on the contacting metal surface due to the presence of a dense hydrogen bonding (H-bonding) network. The lubricating performance of EG-BL outperforms EG-SL, which could be attributed to the denser H-bonding sites in BL and its broader molecular weight distribution. The disc wear loss of EG-44BL is only 45.7% of that lubricated by EG-44SL. Overall, H-bonding is the major contributor to the different tribological properties of BL and SL in EG-based lubricants. Lignin, a cross-linked polymer with phenylpropanoic monomers, is the second most abundant biopolymer in nature . It holdIn addition, the structure and chemical composition of lignin vary in different plant species, or while using different extraction processes and subsequent treatments, thus increasing the complexity of lignin processing and decreasing its applicability in industrial processes ,7. In gep-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol), as shown in According to the literature to date, lignin could be isolated from a wide range of plant biomass resources including but not limited to wood, grass, and bamboo ,15,16. TLubricants have been essential to human activity, with their purpose gradually shifting from “mobility” in ancient eras to “durability” and “energy efficiency” in modern times . ConsideRecently, lignin was demonstrated as an effective lubricant additive in ionic liquids , base oiwM and nM than that of BL. In addition, the PDI of BL is larger than that of SL, which indicates that BL has a broader molecular weight distribution.The average molecular weight of lignin is an important performance index when lignin is used as a lubricant additive. p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol [Lignin mainly consists of three different cinnamyl alcohol monomers, i.e.,  alcohol ,34. The  alcohol . Concern−1, representing the O–H, C–H, and C–O stretching bands [The IR spectra in ng bands . This shng bands ,37. It cng bands . The O–HThe thermal stabilities of EG-based lubricants were further characterized. Since viscosity is closely related to the tribological properties of lubricants, it was measured and is summarized in The ball wear diameter is another important indicator of anti-wear properties. With a reduced disc wear volume achieved by adding 17 and 29 wt% lignin in EG, an enlarged ball wear diameter was surprisingly observed, see Picea abies) and silver birch (Betula pendula) chips. The detailed preparation method is described in our previous publication [Ethylene glycol  was purchased from Sigma Aldrich . Spruce and birch lignins were prepared by the organosolv pretreatment method from Norway spruce  was added to EG at room temperature and then heated at 140 °C in a 150W microwave processor  with magnetic stirring for 10 min. After the microwave processing, homogeneous solutions were formed and denoted as EG-17SL, EG-29SL, EG-38SL EG-44SL, and EG-50SL. The same procedure was applied to dissolve birch organosolv lignin in EG and the prepared mixtures were named EG-17BL, EG-29BL, EG-38BL EG-44BL, and EG-50BL, respectively.2 atmosphere from 20 to 600 °C with a heating rate of 10 °C/min. Fourier transform infrared-attenuated total reflection (FT-IR-ATR) spectra were recorded with a Thermo Scientific Nicolet 380 series spectrometer . The lubricant viscosity was reported within a shear rate range of 1~100 s−1 using a Bohlin CVO 100 rheometer  at 25 °C. A cone-on-plate geometry was used with a 1° cone angle and 20-mm cone diameter. The lower plate had a diameter of 60 mm. The thermal stability of pure EG and their mixtures with spruce or birch lignin was determined by thermogravimetric analysis  in NaR): 20 nm) slides under reciprocating motion against a stationary steel disc : 120 nm). The disc was supplied by Optimol Instruments Prüftechnik GmbH, Germany. The ball was provided by SKF, Göteborg, Sweden. Before each test, the device and sample were cleaned with acetone and ethanol, followed by a uniform application of 0.5 mL lubricant on the steel disc using a glass rod. All tests were conducted under a load of 150 N (2.5 GPa Maximum Hertzian pressure) at 25 °C, a sliding frequency of 50 Hz, and an amplitude of 1.0 mm. The friction coefficient curves were recorded automatically with a data acquisition system linked to the SRV-III tester. After the tests, the wear volumes of the lower discs and wear diameters of the higher balls were determined using an optical profiling system (Zygo 7300). Three duplicate friction and wear tests were carried out to minimize the experimental error.An Optimol SRV-III oscillating friction and wear tester was used to evaluate the tribological properties of the prepared lubricants based on ASTM D 6425 protocol. During the test, the upper steel ball (52,100 bearing steel, diameter: 10 mm, surface roughness (Organosolv lignin dissolution was dissolved in EG up to 50 wt%. The addition of organosolv lignin in EG results in outstanding lubricating properties. The enhanced anti-wear property of the EG/lignin system could be attributed to its excellent adhesion ability on the metal surface and superior lubrication film strength. Specifically, the wear volume of the disc lubricated by EG-44BL is only 8.9% of that lubricated by pure EG. The viscosity difference between EG-BL and EG-SL at different lignin loadings depends on the balance between the thickening effect of lignin itself and the H-bonding between lignin and EG. The lubrication property of EG-BL exceeds that of EG-SL, which could be attributed to the denser H-bonding sites in the molecular structure of BL as well as its broader molecular weight distribution. This work offers a new avenue of utilizing bio-products from different sources in advanced tribological lubrication systems.


Diabetes mellitus (DM) is a widespread metabolic disorder worldwide. Clinical physicians have found diabetic patients have mild to middle cognitive dysfunction and an alteration of brain monoaminergic function. This study explored the change in various patterns of behavioral models and brain monoamine function under streptozotocin (STZ)-induced type 1 diabetes.We established a type 1 DM model via intravenous injection with STZ (65 mg/kg) in rats. Three weeks after the STZ injection, various behavioral measurements including the inhibitory avoidance test, active avoidance test and Morris water maze were conducted. Finally, all rats were dissected and the concentrations of monoamines and their metabolites in cortex and hippocampus were measured by high performance liquid chromatography with electrochemical detection.We found that STZ induced type 1 diabetes (hyperglycemia and lack of insulin) in rats. STZ-induced diabetic rats had cognitive impairment in acquisition sessions and long-term retention of the active avoidance test. STZ-induced diabetic rats also had cognitive impairment in spatial learning, reference and working memory of the Morris water maze. STZ significantly reduced concentrations of norepinephrine (NE) in the cortex and dopamine (DA) in the hippocampus, but increased concentrations of DA and serotonin (5-HT) in the cortex 35 days after injection. The concentration of 5-HT in the hippocampus was also significantly increased.The data suggested that this cognitive impairment after a short-term period of STZ injection might be related to cortical NE dysfunction, differential alteration of cortical and hippocampal DA function, and brain 5-HT hyperfunction. The prevalence of DM in adults was 4.7% in 1980 and increased by up to 8.5% in 2014. The hyperglycemia was resulted from either deficient levels of insulin (type 1 DM) or from defective insulin action (type 2 DM). Hyperglycemia can trigger diabetic ketoacidosis, causing a life-threatening impact in acute complications. Over time hyperglycemia may damage the heart, blood vessels, eyes, kidneys and nerves. Thus, DM can lead to many chronic complications including heart attack and stroke (cardiopathology), kidney failure (renopathology), leg amputation , vision loss (retinopathology), and nerve damage  -6. Thesetentials . There itentials , 12. Howtentials , 14.Streptomyces achromogenes, produces pancreatic islet β-cell destruction and is widely used to produce the type 1 DM model in rodents  and alloxan often induce hyperglycemia in rodents 15, 16). STZ, an antibiotic isolated from , 16. STZ rodents -23. Howe rodents -27. Myhr rodents . TherefoDrugsStreptozotocin  was dissolved with 0.1 M citrate buffer (pH 4.5). All standards including 4-hydro-3-methoxymandelic acid (VMA), norepinephrine (NE), 3,4-dihyroxyphenethylamine hydrochloride (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxytyramine creatine sulfate (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) were purchased from Sigma-Aldrich Co (USA). Glucose, triethylamine and ortho-phosphoric acid were purchased from Merck (Germany). Sodium 1-octanesulfonate was obtained from Tokyo Kasei Kogyo Co Ltd. (Japan). Ethylenediaminetetraacetic acid disodium salt 2-hydrate (EDTA) and acetonitrile were obtained from JT Baker (USA).Experimental animals°C) and humidity (60%) regulated environment with a 12 hr–12 hr light/dark cycle (light phase: 08:00 to 20:00). The Institutional Animal Care and Use Committee of I-Shou University approved the experimental protocol (IACUC-ISU-9905), and all rats were cared for according to the Guiding Principles for the Care and Use of Laboratory Animals. After one week of acclimatization, the rats were used in the experiments described below.Male Sprague-Dawley rats, weighing 180-230 g, were obtained from the National Laboratory Animal Breeding and Research Center. They were housed in groups of four, chosen at random, in wire-mesh cages (39 cm ×26 cm×21 cm) in a temperature , long-term retention (LTR) of the active avoidance test was evaluated and then all rats were dissected. All behavioral tests were conducted between 09:00 and 17:00. The experimental schedule is shown in The rats of the type 1 DM group (STZ group) were induced by a single intravenous injection of STZ with a dose of 65 mg/kg body weight after overnight fasting . The ratOGTT°C). Plasma was stored at -80 °C and the plasma insulin concentration was determined using a fluorescence radioimmunoassay (FRIA) kit from Abbot (USA).According to the experimental schedule, the glucose and insulin responses of all rats following oral loading with glucose were evaluated. Blood was collected from the tail vein before and 30, 60, 90, and 120 min after oral administration with glucose , and were separated into two parts. One part was used to measure the blood glucose level. The other part was drawn into EDTA-coated Eppendorf tubes, and centrifuged at 3,000 rpm for 10 min . One of the compartments (48×20×30 cm) was equipped with a 20 W lamp located centrally at a height of 30 cm, and the other was a dark compartment of the same size, connected through a guillotine door (5 ×5 cm).nd day after STZ injection), each rat was placed in the light compartment with its back to the guillotine door and then the door was opened. The step-through latency (STL) taken by the rat to enter the dark compartment was measured. Once the rat entered the dark compartment, the door was closed. An inescapable scrambled footshock (0.8 mA for 2 sec) was delivered through the grid floor. The rat was removed from the dark compartment 5 sec after footshock. Then the rat was put back into its home cage until the retention trial, which was carried out 24 hr later. The rat was placed in the light compartment again and as in the case of the training trial, the guillotine door was opened and the STL was recorded and used as a measure of retention. An upper cut-off time of 300 sec was set . The apparatus consisted of a shuttle box with two compartments (48×20×22 cm) with a lamp (4 W) and 1000-Hz 75-dB tone, which was used as the conditioned stimulus (CS) and presented for 10 sec until the rat crossed to the opposite compartment. If the rat did not cross to the opposite compartment, it would receive a scrambled 0.8-mA electric foot-shock through the grid floor for 5 sec . When the rat crossed to the opposite compartment during the CS, an avoidance response was recorded. The training session consisted of 20 training trials for five consecutive days with inter-trial interval of 30 sec. The long-term memory retention (LTR) was tested one week later, also using 20 trials .Non-spatial performance of Morris water maze°C) to a depth of 35 cm. The swimming performance activity of each rat including the latency to find the platform, total distance traveled, time, and distance traveled in each quadrant was monitored using a CCTV camera and analyzed by a video tracking system .To assess visual acuity, spatial performance learning and memory function, the rats were tested in a Morris water maze. The Morris water maze was performed in a circular pool  that was made of stainless-steel and filled with water  was placed into the northeast quadrant of the pool at 1 cm above the water surface. Each rat was given four trials to locate the platform , the probe test was performed , the platform was again submerged 1.0 cm below the water surface, but introduced to the pool in the quadrant diametrically opposite its original position. Each rat was given one trial to acclimatize to find the new set of conditions. After four hours, a second trial was carried out in the same manner, but each rat was placed in another quadrant, and the time and length traveled to find the hidden platform were measured , and centrifuged again at 14,000 rpm for 10 min at 4 °C. The collected samples were stored at -80 °C and the concentrations of the cortical and hippocampal biogenic amines  and their metabolites  were measured using HPLC with ECD. An HPLC Model PM80 , a Data Model M746 (Waters Associates), an electrochemical detector Model LC-4C  and a Bioanalytic system MF-6026 column were used.After the LTR of the active avoidance test, all rats were decapitated, and their brains rapidly removed from the skull. Following the method by Glowinski and Iversen , rat braStatistical analysesP<0.05 in all statistical evaluations.All data are presented as mean±SEM based on six rats in each group. All data including the inhibitory avoidance test, active avoidance test, Morris water maze, plasma glucose and insulin levels as well as the concentrations of cortical and hippocampal biogenic amines were analyzed using the unpaired Student’s t-test. The threshold for statistical significance was OGTTP<0.001).One week after VEH or STZ injection, the glucose levels of the VEH and STZ groups were different. The glucose level of the VEH group was 98.14±2.16 mg/dl, and that of STZ group was 450.75±32.73 mg/dl. The average body weight of the VEH and STZ groups were also different three weeks after injection. The average body weight of the VEH group was 325.00±6.45 g (207.50± 4.79 g before injection), and that of the STZ group was 203.06±12.20 g (212.22±4.49 g before injection). The insulin level of the STZ group was significantly lower than that of the VEH group. Thirty minutes after oral administration with glucose, the plasma glucose and insulin levels of the VEH group were elevated when compared with those before treatment. Then, the plasma glucose and insulin levels of the VEH group gradually declined from 60 min to 120 min after glucose treatment . HoweverInhibitory avoidance test and pain threshold to electric stimulationP>0.05). There was no difference between the STL of the VEH or STZ groups in the retention trial (P>0.05) (P>0.05).Twenty-two and twenty-three days after STZ injection, the STL of the STZ group in the acquisition trial was longer  than that of the VEH group ((P>0.05) . As showActive avoidance testP<0.01) (th day after STZ injection) between the VEH and STZ groups (P<0.01) . There was a significant difference in the avoidance numbers during consecutive 5-day training sessions between the VEH and STZ groups (P<0.01) . There w(P<0.01) .Morris water mazeP>0.05) (P>0.05)  reaching the visible circular platform than those spent by the VEH group (P>0.05) . However(P>0.05) .For the next consecutive three days, the VEH group took a gradually declining time and distance to reach the hidden platform on the spatial performance trials of the Morris water maze. The STZ group spent more time and P<0.05, P<0.01)  .P<0.001) (P<0.01) (In the probe test (33 days after STZ injection), the time spent in the target ring and the length ratio  of the STZ group were significantly and obviously shorter than those of the VEH group (P<0.001) . Thirty-(P<0.01) .Cortical and hippocampal monoamine levelsP<0.05) (P<0.05)  . STZ als(P<0.05) .via IV injection with STZ (65 mg/kg) in rats. STZ caused hyperglycemia, hypoinsulinemia and some diabetic syndromes such as polyuria and polydipsia. There was a higher, but slower uptrend in the blood glucose levels of STZ-diabetic rats than those of normal rats in the OGTT. This phenomenon might be due to increased insulin secretion after the oral glucose treatment in normal rats, and the lack of insulin secretion in STZ-diabetic rats.Clinical physicians have indicated that DM patients had mild to moderate cognitive dysfunction compared with healthy controls . STZ is et al.  to induce memory formation. Due to very different results from previous reports regarding the inhibitory avoidance response of STZ-diabetic rodents , 36, 37,et al. , but difet al. , 36, 37.et al. , we furtet al. -41. There.g., running from one side of a two-compartment box to the other when a conditioned stimulus (CS) is presented to escape or avoid the unconditioned stimulus. This paradigm is a relatively complex learning model and is also based on emotional associative learning involving the amygdala, hippocampus, thalamus, and cortex. The present result was consistent with those of Kucukatay et al.  of STZ-diabetic rats with that of normal rats as previous reports have also shown the different results where STZ could impair or facilitate the active avoidance response of STZ-diabetic rodents , 42. TwoFinally, many researchers have demonstrated that STZ impairs spatial performance and reference memory in the Morris water maze from one to six months after induction , 43. TheMyhrer pointed out that cerebral monoaminergic activities strongly influenced learning and memory in four behavioral tasks . Early sTaking all the above observations into consideration, intravenous injection with STZ (65 mg/kg) in rats caused cognitive deficits in some behavioral tasks such as the active avoidance test and Morris water maze but not the passive avoidance test. However, STZ did not alter pain threshold, visual acuity, and motor activity within one-month after injection in rats. There were lower cortical NE and hippocampal DA levels in the STZ-diabetic rats than those of the normal rats. However, there were higher cortical and hippocampal 5-HT levels in the STZ-diabetic rats than those of the normal rats. Therefore, the present study suggests that STZ could induce diabetic syndromes and impair the mild cognitive function in rats. The cognitive dysfunction caused by STZ must be dependent on the complexity of behavioral models within one-month after induction and related to the alteration of monoamine neurotransmitter systems, particularly in brain regions such as NE in the cortex, DA in the hippocampus, and 5-HT.


However, differences in collimator use are one of the most important factors that cause variation among institutions and studies in heart-to-mediastinum (H/M) ratio. Therefore, standardization among various gamma camera-collimator combinations is needed. Previously, a phantom has been developed to cross-calibrate different acquisition conditions in Japan. For further cross-calibration of European myocardial 123I-mIBG imaging, the aim of this study was to collect 123I-mIBG data for H/M ratios from common European gamma camera vendors.Planar myocardial 210 experiments were performed in 27 European institutions. Based on these experiments, conversion coefficients for each gamma camera-collimator combination were calculated. An averaged conversion coefficient of 0.88 was used to calculate a standardized H/M ratio.On average, LE-collimator-derived H/M ratios were significantly lower compared to ME-collimator-derived H/M ratios. The mean conversion coefficients ranged from 0.553 to 0.605 for the LE-collimator group and from 0.824 to 0.895 for the ME-collimator group.Clinically established H/M ratios can be converted into standardized H/M ratios using cross-calibrated conversion coefficients. This standardization is important for identifying appropriate thresholds for adequate risk stratification. In addition, this cross-calibration enables comparison between different national and international data. International efforts have been made to harmonize and standardize cardiac 123I-mIBG scintigraphy.123I-mIBG activity (MBq), scanning parameters, and analysis of the acquired data to obtain the most used semi-quantitative parameters .Cardiac 123I emits high-energy photons of 529 keV which penetrate the relatively thin septa of low-energy (LE) collimators. This penetration leads to degradation of image quality and ultimately introduces variation in H/M ratios.123I-mIBG imaging, however, at the expense of spatial resolution.–123I-mIBG scintigraphy because of their wide availability.123I-mIBG scintigraphy-derived parameters and single-centre results could not easily be extrapolated to other institutions.Collimator choice is one of the most important factors causing variation among institutions and studies.123I-mIBG imaging has been developed to cross-calibrate different acquisition conditions.In Japan, a phantom for planar cardiac 123I was mixed with 4450 ml water to fill the phantom. Since all organ parts were connected as one compartment, no radionuclide concentration adjustment was required for each organ separately. A 3 cm acrylic plate was placed over the phantom to simulate human body attenuation, when imaging was performed. The 256 × 256 matrix images were acquired from the anterior and posterior views for 5 minutes, comparable to clinical planar cardiac 123I-mIBG imaging  of 123I for water as 0.147 cm−1. The standard equation for attenuation was used . The mathematical calculated reference H/M ratio was corrected for attenuation, while Compton scatter and septal penetration of gamma rays were not included. The reference H/M ratios determined by the structure of the phantom were 2.60 and 3.50 . Instead of the original phantom used in the Japanese studies, a new light-weight phantom was used in the European study. In the latter phantom, although the dimensions of the phantom were identical, some acrylic parts were made hollow to fill with non-radioactive water. To obtain identical results compared to the original phantom, minor differences in reference values derived from the light-weight phantom were adjusted. The phantom dimensions of the original light-weighted phantom type were measured by CT scan, and the attenuation in the water and acrylics was recalculated. The adjustment of minor difference of phantom design using linear regression line resulted in agreement of conversion coefficients using low energy high resolution (LEHR), low-medium-energy general purpose (LMEGP), and medium energy general purpose (MEGP) collimators.All Ki  was used to convert the institutional H/M ratios to the reference values (H/M ratioref). In the second step, the H/M ratioref was converted to a standardized H/M ratio using the Kstd. This process can be summarized as:In this study, two H/M ratios (anterior and posterior acquisitions) from each institution were plotted against the reference values Figure . A lineaKstd was 0.88, defined as the average K values for typical ME collimators. The rationale for this conversion to the common ME-collimator type is based on the recommendation to use ME collimators.t test. The linear regression equation of the H/M ratios between two conditions was calculated by the least square method. The statistics software JMP  was used and mathematical calculation was based on Mathematica 10 .The data are shown as mean ± standard deviation. Differences among groups were examined by one-way analysis of variance and Student’s 123I-mIBG phantom studies were performed in 27 institutions in Austria, Belgium, the Netherlands, and the United Kingdom including camera vendors of Siemens (n = 148), GE (n = 44) and Philips (n = 18). Collimator types were divided into 2 groups: LE and ME. The LE group included LEHR, general purpose (LEGP), and all-purpose (LEAP) collimators. The ME group included LMEGP, MEGP, and low-penetration (MELP) collimators.210 n = 113)- and ME-collimator (n = 97)-derived H/M ratios were 1.932 ± 0.056 and 2.685 ± 0.088, respectively, (p = <0.0001). Similarly, for the phantom H/M ratio of 3.50, the LE-collimator- and ME-collimator- derived H/M ratios were 2.281 ± 0.074 and 3.354 ± 0.124, respectively, (p < 0.0001)   Figure . Figure p < 0.0001). However, when the conversion coefficients for MEGP and MELP were combined, the difference was no longer statistically significant. In contrast when the conversion coefficients for LEGP and LEAP were combined, the statistical significant difference persisted between the European and Japanese data.Overall, there were no significant differences when the European conversion coefficients of LEHR, LEAP, LMEGP, and MEGP collimators were compared with the Japanese conversion coefficients Table . Only th123I-mIBG cross-calibration phantom study to calculate conversion coefficients for specific individual gamma camera-collimator combinations. The cross-calibration allowed for a conversion of institutional H/M ratios to standardized H/M ratios. These conversion coefficients will facilitate multicentre comparison of myocardial 123I-mIBG results and enable the extrapolation of the outcome of single- and multicentre studies to other institutions.These are the results of the first European myocardial The design of collimator septa and apertures has influence on the septal penetration. ME collimators, as recommend by the EANM Cardiovascular Committee,The present study shows that the conversion coefficients of the LE collimators are lower compared to the ME collimators which is in line with previous phantom studies in Japan Table . The con123I-mIBG compartment between the conventional and light-weight types. Furthermore, one could expect minor differences between Japanese and European camera combinations due to differences in the design of the collimator septa and apertures and gamma camera crystals. However, we have confirmed with the manufacturers that both collimators and gamma cameras used are manufactured identical for Europe and Japan. The differences between LEAP and MELP may also be explained by small variations in acquisition. In Europe and Japan, both energy windows of 15% and 20% have been used according to local protocol. The acquisition time ranged from 3 to 10 minutes in Japan and was 5 minutes in Europe. The distance from collimator to phantom was the same in Europe and Japan. Moreover, although 123I was manufactured by different companies in Japan  and Europe , both products showed no contamination of other isotopes. Finally, there might be a difference between used acrylics of the original phantom and water in some compartments of the light-weight phantom. Although both water and the used acrylics have an almost identical decay coefficient with a very similar scatter pattern, this minor difference in phantom design may explain the found differences in coefficient values. In summary although there is no variation between Europe and Japan for most LE and ME groups, there is a minimal difference in the LEAP and MELP collimator group. As shown, there is a variety of possible explanations for this small difference. However, except for the relative small number of experiments, the above mentioned factors are true for all comparisons between the European and Japanese data. Hence, if valid, these factors would have caused also differences between the other collimators groups. Therefore, this difference is most likely driven for the largest part by the relative small number of experiments. Of course variation in phantom design, variation in energy windows, and variation in acquisition time may have also contributed.There are several factors that could explain variation in the LEGP and MELP sub-group between the European and Japanese institutions. Most likely, the small number of LEGP and MELP collimators may have resulted in a limited statistical power. In addition, these differences may be explained by small differences in the phantom used. In contrast to the original designed phantom used in Japan, a light-weight phantom was used for the current European study. After careful examination of both cross-calibration phantoms by CT scanner , under the same conditions (120 mAs and 130 kV), there was a small difference of <1 mm of the Our study has some limitations. Compared to the Japanese cross-calibration study, we only used 2 (2.60 and 3.50) instead of 4  references H/M ratios. However, conversion coefficients from 2 data points were nearly identical to those 4 data points.123I-mIBG scintigraphy.In conclusion, differences in gamma camera-collimator combinations can be corrected to standardize ME-collimator values with the use of a cross-calibration phantom. This method can readily be applied, reducing variation in outcome measures and thereby further the clinical role of myocardial 123I-mIBG studies previously published. In addition, future multicentre studies should aim for the use of a standardized H/M ratio, overcoming the impact of gamma camera and collimator differences. Finally, to further stress the importance of standardized H/M ratios, standardized values are essential in risk models for cardiac mortality.Standardization of H/M ratios has impact on patient management. Most importantly, standardization of H/M ratio allows for the development of a universal prognostic threshold. This could be established by reanalyzing databases from a number of


Background: We proposed to verify the role of growth hormone receptor gene expression in growth failure of children with Crohn's disease (CD).Methods: We measured serum levels of growth hormone binding protein (GHBP) and insulin-like growth factor-I (IGF-I), and growth hormone receptor (GHR) gene expression in peripheral blood mononuclear cells of 21 patients with CD (before and after therapy) and in 27 age-sex-matched controls.Results: At diagnosis, significantly lower insulin-like growth factor-I and growth hormone binding protein levels were found in the CD group compared to the controls. Growth hormone receptor mRNA expression was lower in patients at diagnosis compared to the controls, even though the difference did not reach statistical significance, and significantly increased in patients in the following year. Insulin-like growth factor-I levels showed significant improvements 1 year after diagnosis compared to basal levels. On the contrary, growth hormone binding protein values had not significantly changed after 1 year of therapy.Conclusion: Our study raises the hypothesis of another mechanism through which cytokines interact with the growth hormone/insulin-like growth factor-I (GH/IGF-I) axis. Crohn's disease (CD) is a chronic inflammatory bowel disease (IBD) that can affect the entire gastrointestinal tract . AlthougMalnutrition and impaired growth are the major complications of pediatric CD  and apprThe GH-IGF I axis is the major regulator of longitudinal bone growth: the systemic actions of the growth hormone (GH) are mediated by IGF-I, which is produced by the liver. Furthermore, GH has been demonstrated to act, at the epiphyseal growth plate, either directly by stimulating resting/stem-like chondrocytes proliferation, or indirectly, through IGF-I, to promote chondrocyte hypertrophy . Levels Since the process of growth is supported by an adequate supply of nutrients, chronic malnutrition has long been implicated as a cause of poor growth in CD patients . OptimizTo verify whether growth hormone receptor (GHR) gene expression plays a role in the growth failure of children with CD, as a result of the chronic inflammatory condition and malnutrition, we measured GH binding protein serum levels and GHR gene expression in lymphocytes from peripheral blood of patients with CD and from age-sex-matched controls. In CD patients, we analyzed the same parameters 1 year after onset of the disease and correlated them with anthropometric data and clinical activity, in order to discover whether these parameters may be influenced by therapy.In this multicenter study, 21 patients  with a mean age of 12.1 ± 0.9 years were enrolled at the Bambino Gesù Children's Hospital in Rome and at the Pediatric Gastroenterology Division Unit of the University of Naples hospital. Subjects were consecutively recruited over 12 months and were evaluated prospectively for 1 year. Diagnosis of CD was based on clinical history, physical examination, radiologic studies, endoscopic appearance, and histological findings, according to the revised Porto criteria . PatientAnthropometric measurements were taken by a professional nurse and recorded in the clinical diary, in accordance with World Health Organization guidelines. Stature was measured with a stadiometer in patients standing barefoot. Height was expressed according to chronological age, as a standard deviation score.Patients had both elevated levels of both C reactive protein  and of erythrocyte sedimentation rate  at disease onset. Range of CRP and ESR variations are showed in Table The common presenting symptoms were abdominal pain (6/21), diarrhea (11/21), weight loss (9/21), malnutrition (5/21), rectorrhagia (5/21), and extraintestinal manifestations . Perianal lesions were present in 4/21 patients .Pediatric Crohn's Disease Activity Index (PCDAI) was determined according to Valletta et al. , with clBlood was collected on the same day as the baseline clinical evaluation and after 1 year of therapy to measure IGF-I, GHBP and GH-R gene expression. Regarding therapy, at diagnosis patients were prescribed: mesalazine , azathioprine , enteral nutrition , oral steroids , nutritional treatment , nine-month infliximab therapy  and 6-month adalimumab therapy .At follow up, a decrease was found in both CRP and ESR . At this time, the average PCDAI score was 6.5.Twenty-seven healthy children, sex- and age-matched (mean age: 9.4 ± 0.27 years), were recruited as a control group at the Pediatric Auxology Unit of the Fondazione IRCCS Policlinico San Matteo Hospital. Their mean height was −0.66 ± 0.03 SDS and their mean Body Mass Index (BMI) was −0.8 ± 0.19 SDS .Written informed consent was obtained from parents or legal guardians of all children, and patients older than 13 signed a statement of assent. The study was approved by the Ethics Committee of the centers involved in the study.Serum levels of GHBP were measured by a commercially available ELISA . The minimum detectable concentration was 1.69 pmol/l. The intra- and inter-assay coefficients of variation were 5.59–4.78% and 8.36–5.11%, with a quality control range of 20.25–198.24 pmol/l and 19.99–195.78 pmol/l, respectively.The serum IGF-I concentration was measured by an automatic assay that utilizes a solid-phase, enzyme-labeled chemiluminescent immunometric assay . The intra-assay coefficients of variation were 3.9–2.4%, with a quality control range of 77–1,358 ng/ml. IGF-I values are expressed as mean a standard deviation score (SDS) according to Elmlinger et al. .Peripheral blood mononuclear cells (PBMC) of patients and age-matched controls were separated by Ficoll density gradient centrifugation using a standard procedure .2, 0.1 M DTT, and RNAase OUT, was prepared for each sample. The reaction mixture was then added to the RNA/primer mixture, samples were mixed briefly and kept at room temperature for 2 min. Fifty units of SuperScript II RT was added to each tube, the samples were mixed and incubated at for 10 mins, and the tubes were then incubated at for 50 mins, heat inactivated at for 15 mins, and chilled on ice. First strand cDNA was stored at −20°C until use for real-time PCR. Quantitation of GHR mRNA expression was determined by quantitative real-time RT-PCR  and assays on demand were used (Hs00174872_m1 Applied Biosystems). Normalization and validation of the data were carried out using glyceraldehyde-3-phosphate dehydrogenase (GADPH) as a housekeeping control gene. Each GHR or GAPDH probe was labeled with a fluorescent reporter . RT-PCR was carried out with the SuperScript First-Strand Synthesis System. An RNA/primer mixture was prepared containing total RNA, oligo dT (50 ng/μl), 10mM dNTP mix and DEPC water. The samples were incubated at for 5 mins and then on ice for at least 1 min. A master reaction mixture, containing 10X RT buffer, 25 mM MgCl5) attograms GAPDH. We chose the GAPDH gene because its expression shows reproducible and stable levels, moreover GAPDH CT are in the correct range for comparisons with the GHR gene. Furthermore, we evaluated possible variations in GAPDH expression values before considering them suitable for GHR gene expression data normalization and if necessary, disregarded the results.Quantitative Real-time PCR data were calculated by a standard curve and expressed as attograms GHR/; whereas the non-parametric Wilcoxon test for paired samples was used to compare values in patients before and after 1 year of treatment. Correlations were analyzed using the Spearman's rank correlation test.Data are expressed as the mean ± SEM. Statistical differences between patients before therapy and controls were determined using the Mann-Whitney p < 0.05 was considered statistically significant.A value of At recruitment, CD children presented a mean height of −0.55 ± 0.3 SDS Figure  and in tp = 0.007) lower levels of IGF-I were found in the CD group  compared to the control group  lower in patients compared to controls  compared to controls (9515.06 ± 2605.68 agGHR/5 × 105agGAPDH) albeit not statistically significant  increased after 12 months of therapy (−0.045 ± 0.25) compared to the onset of disease  increase in GHR mRNA expression in all patients at 1-year follow-up (37572.17 ± 22135.23 agGHR/5 × 105agGAPDH) in comparison with basal values  improved in the year following diagnosis (−0.051 ± 0.2 SDS) compared to levels at disease onset  family. In particular, cytokines such as Interleukine-6 (IL-6) increased SOCS3 expression, reducing STAT5b activity .We wanted to verify whether GHR gene expression plays a role in the growth failure of children with CD, as a result of the chronic inflammatory condition and malnutrition.In our study, similar to previous reports, the IGF-I level was significantly lower in CD patients compared to controls, consistent with GH resistance . At 1-yeCell surface amounts of GHR are the most important determinant of GH responsiveness . In our To the best of our knowledge, there are no previous studies reporting GHR gene expression values in CD children at onset of the disease and during follow-up. Our study raises the hypothesis of another cytokine interaction with the GH/IGF-I axis. This mechanism involves the GHR before the start of signal transduction. Since GHR is present at growth plate level, it is possible of course that there is not only systemic but also local GH/IGF-I resistance.However, in our study only two patients showed severe growth retardation at diagnosis with a height under −2 SDS (9.5%) and this value is lower than the rate that has been previously reported . This isOur study has some limitations. Firstly, we acknowledge that the sample size was small, secondly the heterogeneity in drug treatments. Further studies with a larger sample size may provide more precise results.Serum IGF and GHR gene expression levels change during the course of therapy, according to the clinical features, suggesting that disease activity modification is accompanied by variations in hypothalamic/pituitary-related factors. During disease remission, IGF-I and GHR mRNA expression increase, supporting the possibility of a strong correlation between growth factors, inflammatory process and nutritional status. These findings underline the importance of prompt control of inflammatory activity in order to prevent stunted growth and to promote good overall outcomes in mild to moderate CD. Furthermore, monitoring CD patients' growth is indispensable in order to assess their response to therapy over time .The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, on request of any qualified researcher.Written informed consent was obtained from parents or legal guardians of all children, in accordance with the Declaration of Helsinki; patients older than 13 years signed a statement of assent. The study was approved by the Ethics Committee at both the Bambino Gesù Hospital and San Matteo Foundation Hospital.MB and PD designed the study, revised the manuscript, and approved its final version. SP and CM performed experiments, collected, and analyzed data. SP and EB wrote the manuscript. CS, EM, and MM gave technical support and conceptual advice. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


S)-Protopanaxadiol (PPD), a bioactive compound extracted from ginseng, possesses cardioprotective, neuroprotective, anti-inflammatory, antiestrogenic, anticancer and anxiolytic effects. However, the clinical application of PPD is limited by its weak aqueous solubility. In this study, we optimized an efficient method of preparing its phospholipid complex (PPD-PLC) using a central composite design and response surface analysis. The prepared PPD-PLC was characterized by differential scanning calorimetric, powder X-ray diffraction, Fourier-transformed infrared spectroscopy and nuclear magnetic resonance analyses associated with molecular docking calculation. The equilibrium solubility of PPD-PLC in water and n-octanol increased 6.53- and 1.53-times, respectively. Afterwards, using PPD-PLC as the intermediate, the PPD-PLC-loaded dry suspension (PPD-PLC-SU) was prepared with our previous method. In vitro evaluations were conducted on PPD-PLC and PPD-PLC-SU, including dissolution behaviors and stability properties under different conditions. Results of in vitro dissolution behavior revealed the improved dissolution extents and rates of PPD-PLC and PPD-PLC-SU (p < 0.05). Results of the formulation stability investigation also exposed the better stability of PPD-PLC-SU compared with free PPD. Therefore, phospholipid complex technology is a useful formulation strategy for BCS II drugs, as it could effectively improve their hydrophilicity and lipophilicity.20( S)-Protopanaxadiol  in the gastrointestinal tract through a series of deglycosylation steps [Ginseng, one of the most popular phytomedicines, has been widely used in medical practice for thousands of years in East Asia. Ginsenosides are reported to be the major pharmacologically-active components of ginseng ,2,3,4. 2iol PPD; , a majoron steps . PPD hason steps ,7, neuroon steps , anti-inon steps , antieston steps ,11, antion steps , prostaton steps ,14, ovaron steps  and malion steps ) and anxon steps .However, PPD is a chemical Biopharmaceutics Classification System (BCS) II drug with inferior water solubility and favorable permeability. Our previous study on the properties of free PPD also revealed its relatively favorable oil/water partition coefficient . MoreoveIn this pharmaceutical study, we predominantly focused on a phospholipid complex-based drug delivery system ,32,33,34Therefore, this study has optimized an efficient method to prepare a PPD-loaded phospholipid complex (PPD-PLC), using a central composite design (CCD) and response surface analysis (RSA). The produced PPD-PLC was characterized with differential scanning calorimetric (DSC), powder X-ray diffraction (PXRD), Fourier-transformed infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR) analyses, associated with molecular docking calculation. Equilibrium solubility was determined to compare the performance of PPD and PPD-PLC in hydrophilicity and lipophilicity. Afterwards, by using PPD-PLC as the intermediate, the PPD-PLC-loaded dry suspension (PPD-PLC-SU) was prepared using our previous method . In vitrR2 = 0.8279) was superior to the linear model (Equation (1), R2 = 0.5716) in terms of correlation coefficient (R2). Consequently, the quadratic model was selected as the optimal fitting model and was further validated using an F-test for the analysis of variance (ANOVA) to evaluate the significance of the regression .Entrapment efficiency  of PLC refers to the percentage of the entrapped drugs in the total added drugs when preparing PLC. After the scheduled experiments of CCD, the EE of the 20 batches is shown in gression , which rX1 was found to be favorable to gain high EE. An increase in the X3 level caused the EE to increase initially and to decrease subsequently. However, the level of 2X was observed to have little effect on the variation of EE. In the largest area of the Y value  formed by the three-dimensional effects of response surfaces, the optimal process parameters were obtained as follows: phospholipid-to-drug ratio  = 2:1 and reaction time  = 5 h. The reaction concentration of PPD  was set to 20 mg/mL to increase drug load capacity.The relationship between variables and the response could be visualized using response surface analysis RSA in Design-Expert, which generated the response surface and contour plots. As shown in X1, X2 and X3 were set to 2:1, 20 mg/mL and 5 h, respectively) to evaluate the optimization capability of the generated models derived from the circumscribed CCD and RSD results. The entrapment efficiency obtained with the predicted model was 78.04%, whereas that in PPD-PLC prepared in optimized conditions was 65.94% ± 4.02% (n = 3), with a −15.5% bias.PPD-PLC was prepared under the optimized described parameters (y = 12.9x + 96.6 (r2 = 0.9999). The analytes showed linear behavior in the concentration range of 15.2–1015.6 μg/mL. The limit of detection (LOD) and the limit of quantification (LOQ) were determined by the signal-to-noise method to be 0.3 (S/N = 3) and 1.0 μg/mL (S/N = 10), respectively. The average recoveries were 98.2%, 100.4% and 96.0%  at the three spike levels of 120%, 100% and 80%, respectively. The results of precision with the standards at different levels  validated a good precision in intra-day  and inter-day  investigations. All of these results conformed to the standard recommended by ICH  [Drug loading  refers to the percentage of the drugs in PLC. In this study, EE and DL were determined by detecting the content of PPD in the complex products of PPD-PLC. Method validation included assessments of linearity, sensitivity, accuracy and precision. The linearity investigation results showed that the regression equation of calibration curves was man Use) . TherefoDSC analysis, a convenient method to investigate compatibility, provided information on the interaction between drug and excipients. The DSC thermograms of free PPD, phospholipids, PPD-PLC and the physical mixture of PPD and phospholipids are shown in PXRD is often used to evaluate the existing form of PPD in the PPD-PLC. 3, υ), 1186.22, 1120.64, 1029.99  and 648.08 cm−1 . The wide absorption peaks at 3238.48 cm−1 suggested the formation of hydrogen bonds between molecules caused by the hydroxyl groups. The characteristic absorptions of phospholipids were observed at 2922.16, 2852.72, 1737.86 , 1467.83 , 1242.16 , 1091.71 , 970.19  and 823.60 cm−1 , which correspondingly indicated the presence of saturated alkyl, ester, phosphate and methyl groups. The spectrum of the physical mixture was the superposition of functional group peaks from the two compounds with the same peak positions. In the spectrum of PPD-PLC, the wide peak at 3238.48 cm−1 was shifted to 3207.62 cm−1 with a weakened intensity. The stretching vibration absorption peaks at 1186.22 and 1120.64 cm−1  disappeared, which illustrated that the hydroxyl groups might be influenced by the polar groups. C–H stretching vibration from the alkyl group only appeared at 2924.09 and 2852.72 cm−1.As shown in The spectra of the samples shown in −1.The molecular modeling method can explore the mechanism of interaction on a molecular level. The best interaction sites of PPD and phospholipid could be seen in a full range via molecular docking. After the molecular models of PPD and phospholipid  were estn-octanol (Co) (n-octanol (398.45 ± 1.20 μg/mL) was 1.53-times that of free PPD (261.15 ± 0.35 μg/mL). This strongly suggested that the phospholipid complex could significantly promote the hydrophilicity (p < 0.01) and lipophilicity (p < 0.05) of the free drug.Equilibrium solubility studies of free PPD and PPD-PLC were conducted in water (Cw) and in nol (Co) . The aveThe dissolution curves of free PPD, PPD-PLC and PPD-PLC-SU are shown in In our previous study , the staDuring the property investigation of PPD in our previous research , the sto1H-NMR spectroscopy highlighted that new compounds were not produced in the complex process. The results of molecular docking studies also indicated that the formation of the complex was an exothermic process, which was consistent with the DSC results. The two portions of hydrophobic and hydrophilic interactions matched the two new endothermal peaks, which appeared at 153.67 and 294.16 °C. −1 was shifted to 3207.62 cm−1 with a weakened intensity. At the same time, the formation of intermolecular H-bond also affected the H-bond interaction between the two adjacent –OH groups in PPD molecule. This was also shown in the FTIR data at the wavenumbers of 3238.48 cm−1 and 1120.64 cm−1, where the peaks had disappeared.A series of physicochemical methods were applied to characterize PLC formation. Similar to the observation of previous researchers in their cases of the PLC study , PPD andw/w), with a relatively low purity. As the analytical certificate reported, there was about another 17% “phosphatidylethanolamine” in this phospholipid. Therefore, in our opinion, this diffraction peak might be related with the existence of phosphatidylethanolamine. Some reports in recent years confirmed this inference, in which their results also showed that there were some tiny diffraction peaks in the XRD patterns of free phospholipids, when lecithin of relatively low content (<90%) of phosphatidylcholine was used. This phospholipid materials included Lipoid S75 [Actually, a diffraction peak at about 20(2θ) could be observed in the PXRD pattern of the phospholipid B, althoupoid S75  and Lipopoid S75 . Furtherpoid S75 ,55 , which indicated that the amphiphilic phospholipid molecules might promote the dissolution of PPD. The hydrotropic effects were greatly magnified by the entrapment of PPD into PLC. Much of these amplification effects might be attributed to the superior dispersibility, amorphous form of the phospholipids complex and the combination of polar groups from PPD and excipients. We speculated that the hydrophobic functional group of PPD was close to the phospholipid molecules through quasi-stable bonding, and therefore, the nonpolar moiety of PPD was masked by the phospholipids, which was consistent with another study [w/w) phosphatidylcholine content, which was demonstrated to be biologically safe, non-carcinogenic and non-mutagenic [Using PPD-PLC as the intermediate, PPD-PLC-SU was prepared with proper pharmaceutical formulation, and a dissolution study and storage stability investigations were then conducted. The results of the dissolution study revealed the significantly improved dissolution extent and rates  and 15.83% Poloxamer 188 (wetting agent). This result also confirmed the advantageous property of the dry suspensions formulation, as mentioned above. It can be expected that during the beginning of dissolution test, the PPD-PLC-SU powder was rapidly wetted because of the existence of wetting agent (Poloxamer 188), compared with the PPD-PLC powder. In addition, with the impeller agitation speed of 100 rpm, PPD-PLC-SU was well dispersed in the dissolution media and could be rapidly suspended with the assistance of the suspending agent (Avicel CL611). By contrast, although PPD-PLC also had improved dissolution, compared with free PPD powder, it did not have enough motive force to be moistened and suspended in the dissolution media within a short time. Therefore, PPD-PLC-SU presented more rapid dissolution, when compared with PPD-PLC. The patent on this formulation of the dry suspension was authorized by the Chinese State Intellectual Property Office in the year 2014 (ZL201210120958.5/CN102631322B). The combined application of two or more pharmaceutical technologies was observed to compound the effects. Other PLC-based dosage forms include nanoparticles ,60, SMEDw/w) was purchased from Shanghai Advanced Vehicle Technology Corporation, Shanghai, China. Avicel CL-611 was purchased from FMC Corporation, Philadelphia, PA, USA. Poloxamer 188 (Pluronic F68) was purchased from BASF Corporation, Ludwigshafen, Germany. All other chemical reagents were of analytical grade or higher.PPD with a purity of ≥98% was purchased from Jingke Chemistry Technology Co., Ltd., Shanghai, China. Phospholipid (PL-100M) with a phosphatidylcholine content of 80% , concentration of PPD in the reaction medium  and reaction time  were selected. An optimization procedure was performed by applying a CCD and RSA to systematically analyze the compound influence of these factors in a reduced number of trials. Briefly, in this design, three factors with five levels each were evaluated, and the trials were performed in all 20 possible combinations. EE was considered as the response variable. A statistical model that incorporated the linear effects (Equation (3)) and polynomial terms (Equation (4)) by using Design-Expert was used to evaluate the response. The models [Y is the dependent variable , a0 and b0 are the intercepts representing the arithmetic mean response of the 20 runs and ia, ib are the estimated coefficients for the factor iX. The main effects  represent the average results of changing one factor at a time from its minimum to maximum value. The interaction terms  revealed the response changes when two factors are changed simultaneously. The polynomial terms  were included to investigate nonlinearity. The levels of the three independent variables and the composition of the CCD batches are presented in The process of PPD-PLC was optimized by using a CCD and RSA. According to the results of early preliminary experiments, the factors (independent variables) that had a relatively strong influence on the EE (dependent variables), phospholipid-to-drug ratio .When a certain amount of prepared PPD-PLC was dispersed in 5 mL of ethyl acetate, both PPD-PLC and phospholipids were easily soluble in ethyl acetate; however, the free PPD materials remained essentially insoluble in ethyl acetate. Because of this solubility difference, methanol (a good solvent of PPD) was added to the precipitate (the unentrapped PPD), and the sample was centrifuged. The amount of free PPD was obtained by detecting the content of PPD in the resultant supernatant. Each experiment was performed in triplicate. The EE of PPD-PLC was determined using Equation (6).Approximately 50 mg of PPD-PLC powder were dissolved in 50 mL of methanol. After appropriate dilution, a 20-μL aliquot of the sample solution was injected into a high performance liquid chromatography (HPLC) system after filtration through a 0.45-μm membrane to determine the concentration of PPD in PPD-PLC. Subsequently, the amount of PPD in the samples was calculated. Each experiment was performed in triplicate. The DL of PPD-PLC was determined using Equation (7).The content of PPD in the phospholipid complex was determined with HPLC . Approxiy) and concentration (x). The determination coefficient (r) and linear dynamic ranges were determined. The LOD and LOQ were determined for sensitivity using the signal-to-noise method and expressed by the determinations at certain signal-to-noise ratios (S/N = 3 and 10), respectively. The accuracy of the method was evaluated though recovery experiments by spiking standard PPD into PPD-PLC samples at three levels , with three replicates at each level (n = 3). The mixture samples were then treated with the aforementioned procedure and injected to an HPLC analyzer. The recovery was calculated as the ratio (%) of the determined spiked amount to the actual addition amount of PPD. In addition, the intra-day (n = 6) and inter-day (n = 5) precision studies for three standards  were investigated, respectively, where the results were expressed by the relative standard deviation (RSD).Method validation included linearity, sensitivity, accuracy and precision. The linearity of the calibration curve was established by plotting the detected response area ratio versus the concentration of the standard solutions of different levels. It was established based on the injections of the standard solutions at concentrations within the range of 15.2–1015.6 μg/mL . Each of the solutions was injected in triplicate. A calibration curve equation was calculated for quantification of PPD using the peak area  from 25–400 °C. The peak transition onset temperatures of the samples  were determined using a differential scanning calorimeter . The free PPD, phospholipids and their physical mixture were used as controls.The PXRD pattern of samples  were acquired by increasing the diffraction angle (2θ) from 4°–40° at a scan rate of 0.04° (2θ/s) on an X-ray diffractogram  to investigate the physical state of the drug in PPD-PLC. The free PPD, phospholipids and their physical mixture were used as controls.−1–400 cm−1 region. The free PPD, phospholipids and their physical mixture were used as controls.The samples were taken in KBr pellets using a Fourier-transformed infrared spectrophotometer . The spectra of the samples  were recorded in the 4000 cm1H-NMR spectrometer  at 400 MHz. The free PPD, phospholipids and their physical mixture were used as controls.The samples  were dissolved in deuterium-substituted solvent and analyzed using a O-β-glucopyranosyl-20(S)-protopanaxadiol dehydrate obtained from the Cambridge Structural Database (Refcode: JEPJEU) and reconstructed. The molecular structure of the phospholipid was extracted from the crystal structure (PDB ID: 3B7Q) of yeast sec14 homolog sfh1 in complex with phosphatidylcholine of the RCSB PDB  [The molecular structure of PPD was extracted from the single crystal structure of 20-ta Bank) . The molta Bank)  was usedBefore docking, the energy minimization protocol was executed to establish PPD and phospholipid models. The heavy atom of PPD with a highly rigid cyclic-structure was fixed. A flexible phospholipid molecule was then docked to it. This initial docking result with relatively low energy was used for the subsequent structure optimization.In the minimization protocol, all of the models were put in the vacuum. The non-bond cutoff distance of 18.5 Å, spline width of 1.0 Å and buffer width of 0.5 Å were set. In the docking protocol, a Lamarckian genetic algorithm (LGA) in combination with a grid-based energy evaluation method was used for pre-calculating grid maps, according to the interatomic potentials of all atom types presented in the host and guest molecules, including the Lennard–Jones potentials for van der Waals interactions and Coulomb potentials for electrostatic interactions. A grid map of dimensions (50 Å × 50 Å × 50 Å), with a grid spacing of 0.375 Å, was placed to cover the PPD structure. With the help of AutoDockTools , the atomic partial charges were calculated by the Gasteiger–Marsili method . The parn-octanol in sealed glass containers at 25 °C. Each experiment was performed in triplicate. The liquid samples were agitated for 24 h and then centrifuged to remove excess PPD at 4000 r/m for 10 min. The supernatant was filtrated through a 0.45-μm membrane, and a 20-μL aliquot of the filtrate was injected into an HPLC system to be detected at 203 nm to determine the concentration of PPD.Equilibrium solubility was determined by using the supersaturated solution of free PPD and PPD-PLC in 5 mL of water or The produced PPD-PLC and Poloxamer 188 were ground over a 100-mesh sieve. According to our previous study , the proDissolution studies were performed according to the specifications of the dissolution test apparatus reported . The disThe stabilities of free PPD and PPD-PLC-SU were evaluated by calculating DL upon storage at a high temperature of 60 °C, high humidity (RH 92.5% ± 5%) at 25 °C and 4500 Lx ± 500 Lx illumination conditions. The samples were determined at the 5th and 10th day.t-test was used to compare two samples. Significance was tested at the 0.05 level of probability (p). Data analysis was performed with the SPSS software package  or Design-Expert .All of the data are presented as the mean ± SD or mean. Multiple comparisons of the means (ANOVA) were used to substantiate statistical differences between groups, and Student’s p < 0.05). Molecular docking calculation combined with FTIR and DSC analyses revealed the possible interaction mechanism between PPD and phospholipids, and the mutual reflection of calculation and experiments strengthened the explanation of the mechanism. The results of stability investigations also showed that PPC-PLC possessed superior stability under high temperature, compared with free PPC. Therefore, the phospholipid complex, an efficient intermediate product in dry suspensions, makes PPD a possible candidate drug with great potential for clinical trials and application. Through enhancing the hydrophilicity and lipophilicity of free drugs, the absorption and bioavailability of PPD might be promoted effectively. However, the pharmacokinetic process and in vivo mechanism of PPD-PLC remain to be further investigated.In this study, PPD-PLC was successfully designed and prepared with optimal process parameters, which were developed by applying CCD and RSD. The resultant products were characterized and evaluated to confirm the achievement of favorable physiochemical properties. The results of equilibrium solubility investigations strongly suggested that the phospholipid complex could significantly promote the hydrophilicity and lipophilicity of PPD. The PPD-PLC-SU was prepared with PPD-PLC as an intermediate. In addition, in vitro dissolution behaviors of PPD-PLC and PPD-PLC-SU were also investigated and the results revealed significantly improved dissolution extent and rates (


Objective: Current evidence has revealed a significant association between bullous pemphigoid (BP) and neurological diseases (ND), including stroke, but the incidence of BP autoantibodies in patients with stroke has not previously been investigated. Our study aimed to assess BP antigen-specific antibodies in stroke patients.Design: One hundred patients with stroke and 100 matched healthy controls were randomly selected for measurement of anti-BP180/BP230 IgG autoantibodies by enzyme-linked immunosorbent assay (ELISA), salt-split indirect immunofluorescence (IIF), and immunoblotting against human cutaneous BP180 and BP180-NC16A.Results: Anti-BP180 autoantibodies were found in 14 (14.0%) patients with stroke and 5 (5.0%) of controls by ELISA (p < 0.05). Sera from 13 (13.0%) patients with stroke and 3 (3.0%) controls reacted with 180-kDa proteins from human epidermal extract (p < 0.05). 11 (11.0%) of stroke and 2 (2.0%) of control sera recognized the human recombinant full length BP180 and NC16A (p < 0.05). The anti-BP180-positive patients were significantly younger than the negative patients at the time of stroke (p < 0.001).Conclusion: Development of anti-BP180 autoantibodies occurs at a higher frequency after stroke, suggesting BP180 as a relatively common autoantigen after stroke and providing novel insights into BP pathogenesis in aging. Bullous pemphigoid (BP) is an autoimmune blistering skin disorder most commonly found in the elderly . It is cA significant association between BP and neurological diseases (ND)  has been fully supported by a series of previous studies –9. DevelStroke is one of the most common forms of ND, which often presents as a life-threatening condition and is the second leading cause of death worldwide. It is characterized by an acute onset and considerably severe tissue damage in the brain. Recently, a study including 12,607 patients with first-ever stroke has revealed that 38 (0.3%) patients developed BP in a median of 3.5 years, while only eight people (0.06%) had BP in a median of 3.7 years in the control group . In addiFollowing the principles of the Declaration of Helsinki this study was approved by the Ethical Committee of Peking Union Medical College Hospital and informed consent was obtained from all patients and unaffected individuals. Patients with stroke were from the Department of Neurology, Peking Union Medical College Hospital. These included 82 patients with ischemic, 5 with hemorrhagic, and 13 with both ischemic and hemorrhagic stroke, who had been diagnosed with stroke for 1 week to 19 years before our study. The patients were recruited for review and serum samples were collected during July 2014 to October 2016. Stroke was diagnosed based on medical history, clinical symptoms, and results of neuroimaging in the Department of Neurology. The age- and sex- matched control group was comprised of patients attending the hospital for surgery (benign neoplasms resection) in the Department of General Surgery from 2014 to 2016. The average age of stroke and control patients is 66 years for both populations, with a proportion of male patients of 69 and 74%, respectively. Patients with neurological diseases  and skin diseases  were excluded from the control group after reviewing medical records. Normal human foreskin was obtained from patients receiving circumcision in the Department of Urology.Anti-BP180/BP230 IgG autoantibodies in the sera samples of patients and healthy controls were detected by commercially available ELISAs for human BP180 (NC16A domain) IgG  and BP230 N-and C-terminal domain IgG , according to the manufacturer's instructions .Protein extract preparations, polyacrylamide gel electrophoresis, and immunoblotting were performed as previously described . BrieflyFive micro molar frozen non-fixed sections of human skin (treated with 1 M NaCl) were blocked (1% BSA in PBS) and human sera in 1:4 to 1:320 dilutions were used as primary antibodies. Rabbit anti-human IgG-FITC  was used as the secondary antibody. The skin sections were then mounted with glycerol/PBS  and observed under a fluorescence microscope. A clear linear immunostaining on the BMZ was considered positive, while no fluorescence was considered negative.t-test and rank sum test.The data involved in the statistical analysis included qualitative analysis, such as BP180 antibody (positive/negative), sex , complication (yes/no), and frequency of attack (single/ multiple); and quantitative analysis . Specifically, qualitative data were statistically analyzed by chi square test and logistic regression analysis, and the quantitative data were statistically analyzed by n = 100)  and healthy controls (n = 100) were collected to examine anti-BP180/BP230 IgG antibodies by ELISA . The positive rate of anti-BP180 antibody in the stroke cohort  was significantly higher than that in controls  (P = 0.03) (P = 0.016) (P = 0.018) (P = 0.018) (Sera from patients with stroke ( = 0.03) , 2. All  = 0.03) , 2. Sera= 0.016) . Sera fr= 0.018)  and huma= 0.018) . Anti-BP= 0.018) .The positive rate of anti-BP230 antibodies had no statistical difference between the stroke  and control groups  by ELISA.P = 0.024). The medical records of anti-BP180 positive patients and controls were reviewed and all patients were followed up until October 2017. In the 1-3-year follow-up period, neither stroke patients nor the controls developed BP-like skin lesions. The 1-3-year survival rate of anti BP180 positive patients with stroke and control were both 100%.Anti-BP180 autoantibody titers were significantly higher in the stroke group (19.2 ± 6.07 U/ml) compared to those of control group . Among them, the proportion of patients younger than 60 years in the anti-BP180 positive group  was significantly higher than that of the anti-BP180 negative patients . The duration of follow up after first stroke attack for the BP180 positive group (7.0 ± 2.94 years) was significantly shorter than that of the anti-BP180 negative group . There was no significant difference in sex, complications, and stroke attack times between the two groups  was significantly lower than that of the anti-BP180 negative group  showed binding of IgG autoantibodies to the epidermal side of the BMZ, consistent with works by Messingham et al. , Tuusa eP < 0.001), suggesting that young age might be a risk factor for stroke patients to develop BP. The proportion of patients younger than 60 years in the anti-BP180 positive patients  was significantly higher than that in the anti-BP180 negative patients  (P < 0.001), further supporting that younger stroke patients with shorter duration after first attack are more likely to develop BP antibodies. This may be due to strong immune responses in the early stage after stroke, whereas there is down-regulation of the autoimmune response in the late or recovery stage of stroke  were significantly younger than anti-BP180 negative stroke patients  . The durP = 0.019), while none of them had BP-like lesions . As the anti-BP180 antibodies are correlated with the disease activity of BP, we considered that the difference between the two groups is of clinical significance and may herald the development of BP in the future. Jedlickova et al. found that male stroke patients were more likely to have BP (P > 0.999).Anti-BP180 autoantibody values were significantly higher in the stroke group (19.2 ± 6.07 U/ml) compared to those in the control group by ELISA (12.2 ± 2.39 U/ml;  have BP . In our We conclude that anti-BP180 autoantibodies could be detected at a higher rate in stroke patients than age- and sex- matched controls, supporting that BP180 could serve as a shared autoantigen in both stroke and BP. Our data suggest young age in stroke could be a risk factor for later developing BP and provide a molecular mechanism for BP associated with ND . The preYaW performed most of the experiments, analyzed the data, and wrote the manuscript. LL, BP, and HJ conceived the project and supervised the study. LL, YaW, and DW designed the experiments. XM interpreted data and revised the manuscript. LL, BP, CH, AP, and YiW discussed the results and revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.